Synovial sarcoma (SS) is a common soft tissue malignancy that is routinely associated with a poor prognosis. The initiating event of SS is a single translocation t(X;18) resulting in the fusion of the SS18 gene on chromosome 18 to one of three closely related X-linked genes, termed SSX1, SSX2 and SSX4. A recent study demonstrated that endogenous SS18 and the SS18-SSX fusion protein are subunits of the SWI/SNF (BAF) complex, providing a possible mechanism by which this translocation may perturb gene expression and induce tumorigenesis. Of note, the pluripotency-associated gene Sox2 was recently shown to be expressed in cultured SS cell lines and Sox2 expression was reportedly required for continuous growth in vitro. Furthermore, we have found that Sox2 is upregulated in human SS as well as a mouse model of SS, and that the SS18-SSX-containing BAF complex specifically binds the Sox2 promoter. However, it remains unclear whether Sox2 is required for SS formation in vivo. Moreover, the mechanisms by which Sox2 expression contributes to synovial sarcoma has yet to be determined. Thus, the objective of this R21 application is to dissect the molecular and functional role of Sox2 in SS tumorigenesis in vivo, with the ultimate goal of identifying new therapeutic targets for treatment. We are proposing three independent aims to elucidate Sox2's role in SS biology by combining a preclinical mouse model with human samples. First, we will use transgenic Sox2 reporter and ablation alleles, generated by our lab, to record expression patterns of Sox2 and to assess if Sox2-positive cells act as synovial sarcoma stem cells. Second, we will test whether loss of Sox2 expression in mice is required for tumor formation. Lastly, we will determine genomic targets and binding partners of Sox2 by chromatin immunoprecipitation-sequencing and mass-spectrometry in order to define Sox2-specific transcriptional and signaling networks in synovial sarcoma. We will then use this data to initiate a pilot small molecule screen that targets the gene networks driven by Sox2. Altogether, this exploratory project will contribute to our understanding of Sox2's molecular and functional role in synovial sarcoma and may lead to new strategies to treat this malignancy by targeting stem cells or pathways that trigger differentiation.